The familial process of Amphibian Metamorphosis (Apoptosis) is one of the excellent examples which help to understand the mechanical process and how much it’s normal for each organism. The amphibian body undergoes rapid and dramatic changes to adapt from aquatic to terrestrial life, this condition is known as amphibian metamorphosis. In the larval stage of the frog some physiologic dissimilarities are found from the adults these are tail and gill in the larval stage and forelimb in adults.
During metamorphosis, numerous organs are changed rapidly from the larval stage to adult form. The tail, head, intestine are the major organs which getting change during morphogenesis and form an adult body structure from the larval stage. The thyroid hormone is the key regulatory protein molecule that plays the principal role in the initiation of amphibian metamorphosis. TH can autonomously induce the degeneration of larval specific gill and tail whereas the epithelial apoptosis of the small intestine.
The amphibian metamorphosis is strictly controlled by the cascade TH dependent gene expression which has complex interaction between tissue degeneration of tail, gill as well as intestinal development. Conversion from tadpole to mature frog the maximum changes occur at stage 58-66. The climatic changes that occur at 60-62 staged were the main intestinal changes, as well as the drastic regression of the tail, take place by sires of apoptosis in the larval epithelium and tail muscles at pre-metamorphosis or 57 stages, reach the climax at 58 stages when forelimbs protrude. This total complex regulation automatically controls by TH.
PROTEOLYTIC CASPASES CASCADE: Killing the Cell -Amphibian Metamorphosis (Apoptosis)
Caspase is a cysteine-dependent aspartate protease that contains cysteine at the proteolytic site and works on the c-terminal site of aspartate residues. There are in total 12 different caspases present in cells; those are regulating the apoptosis process from beginning to end. The caspases have different two groups- initiator caspases (caspases 2, 8, 9, 10, 12) and effector caspases (caspases 3, 6, 7). In general, all caspases are present in cells as an inactive condition or as procaspase form. Due to proper stimulation, they are activated and covert to caspase form and induce the process of programmed cell death.
The inactive heterodimeric proteolytic proteins induce amplification of proteolytic cascade activity after activation. The procaspases are responsible for the initiation of proteolytic cascades reaction known as initiator caspases. After activation, the activated initiator caspases activate downstream executioner procaspases which further activate different cellular procaspases, DNases, RNases which promote morphological changes of apoptotic cells. Caspases have the ability to cleave a selected group of cellular essential proteases.
- Various protein kinases like focal adhesion protein kinases (FAK), PKB, PKC, Raf1 have able to disrupt cellular adhesion, cell survival processes. Caspase also hampers the cellular survival by inactivation NF-κβ signaling pathway.
- Caspases induce nuclear condensation by cleaving lamin protein which presents in the inner lining of the nuclear envelope. Due to the cleavage of nuclear lamina shrinkage of the nucleus and disassembly of nuclear lamina occur.
- Caspases also induce structural deformities of cells by cleaving and inactivating the cytoskeletons (microtubule, microfilaments, intermediate filaments).
- An endonuclease called caspase-activated DNase (CAD) activated by caspases and translocate to the nucleus and break DNAs into sequential fragmented DNAs.
Apoptotic Cell Disassembly:
Cellular disassembly is the common character of the apoptotic cells. The cell to be going for apoptosis go through several characteristic morphological changes. There are the most important of the previous hierarchical evets:
- Breaking of cytoskeletons by caspases induces the cell shrinkage, as well as rounding of the cell, which occurs due to the reaction of lamellipodia (figure 2, stage i).
- Cytoplasmic condensation occurs with a thickly packed organization of organelles figure 2: stage ii & iii).
- Chromatin condensation promotes its accumulation at the perinuclear envelope, this event is known as pyknosis, a hallmark of apoptosis.
- The nuclear membrane becomes disappears and fragmented DNAs are seen in the nucleus. Fragmented DNA formation is known as karyorrhexis. The later nucleus breaks into several discrete nucleosomal bodies because of fragmented DNA (figure 2, stage v).
After these events apoptotic cell disassembles are induced in three different steps. These are,
- Bleb formation: Small buds like arrangement form in a membrane known as blebs. These small blebs will rapidly be converting to a larger size and change the dynamic structure of the cell membrane known as membrane blebs (figure 2: stage i-iii). Some regulatory proteins play an important role to control this event.
- Membrane protrusions: In some specific condition a thin, long extension of cell membrane introduce a different morphological change of cell as well as membrane, known as membrane protrusions (figure 2: iv-v).
- Cellular fragmentation as apoptotic body: The degraded components are exposed to macrophage as multiple vesicular arrangements known as the apoptotic body (figure 2: stage v) which undergoes to phagocytosis (figure 2: stage vi). The biochemical changes in cellular membrane and also plasma membrane protrusions induce the phagocytic process.
Removal of Dead Cells:
Removal of apoptotic cells by phagocytes is one of the principal events which help to maintain tissue homeostasis. The series of molecular and cellular events that identified apoptotic cell recognition and uptake. The anti-inflammatory response help to detect and dispose of apoptotic cells at the tissue level, also immunological tolerance. Moreover, under some defined conditions such as killing tumor cells by specific cell death inducers, the recognition of apoptotic tumour cells can increase an immunogenic response and anti-tumor immunity.
During Apoptosis progression apostatized cells exhibit events of cellular and morphologic changes which leads the cellular detachment and organelles fragmentation. These cellular and morphological changes promote phagocytosis by releasing “find me” signals like release soluble factors (nucleotides) or microparticle associated molecules (like CX3C chemokine ligand 1(CX3CL1) and intracellular adhesion moleculw3 (ICAM3) which help to callup phagocytic cells. This total cell clearance gets done by two-phase i. phagocytes recruitment ii. Recognition and engulfment.
- Phagocyte recruitment: This event initiate by releasing soluble factors like fragmented nucleotides and other factors, through caspase-activated pannexine1 (PANX1) membrane channel and promotes the detection of an apoptotic cell by “find me” signal which induces cellular migration of macrophages towards apoptotic cells. The receptor of macrophagic cell-like P2Y2 detects this signal, leads to cell migration. “Find me” signal is very necessary for apoptotic cell engulfment or clearance. The microparticle-associated signals promote the phagocytic cell to preparing for engulfment these signals are induced by CX3C1, ICAM3 molecules that interact with their respective receptors CX3CR1 and ICAMR3. These two major events have occurred in this phase where the apoptotic cell removing started.
- Recognition and engulfment: Phagocytes involve in ‘eat-me’ signals directly by cell surface receptors (like brain-specific angiogenesis inhibitor (BAI-1) and CD91) and also indirectly through bridging molecules (like milk fat globule-EGF factor 8 (MFG-E8)) that are in turn detected by membrane receptors (αVβ3). Subsequent downstream signaling initiates engulfment and engulfment associated responses from phagocytes. Moreover, calreticulin and phosphatidylserine exposure at the outer leaflet of the apoptotic cell membrane as well as the apoptotic body induce “eat me” signal to the phagocytic cell. On the other hand, this signals retriever presence of CD31 which induces “don’t eat me” signals. The formation of apoptotic bodies and microparticles is not fully defined. Rather than this, purinergic receptor P2Y2; ROCK I, Rho-associated coiled-coil containing protein kinase I help to promote actomyosin contraction of cell. Regulation of immune responses and other biological processes also initiates by ligand (CRT) receptor (CD91) interaction of apoptotic cell and phagocytic cells respectively.